Imura Tetsuya, Kornblum Harley I, Sofroniew Michael V
Department of Neurobiology, Brain Research Institute, University of California, Los Angeles, California 90095-1763, USA.
J Neurosci. 2003 Apr 1;23(7):2824-32. doi: 10.1523/JNEUROSCI.23-07-02824.2003.
Periventricular germinal zones (GZs) of developing and adult brain contain neural stem cells (NSCs), the cellular identities and origins of which are not defined completely. We used tissue culture techniques and transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) from the mouse glial fibrillary acid protein (GFAP) promoter to test the hypothesis that certain NSCs express GFAP. To do so, we determined the relative proportions of multipotent neurospheres that are formed by GFAP-expressing cells derived from GZs at different stages of development. In this transgenic model, dividing GFAP-expressing cells are ablated selectively by treatment with the antiviral agent ganciclovir (GCV). Single-cell analysis showed that transgene-derived HSV-TK was present only in GFAP-expressing cells. GCV applied in vitro eliminated growth of multipotent neurospheres from GZs of postnatal and adult transgenic mice but not early embryonic (embryonic day 12.5) transgenic mice. GCV prevented growth of secondary multipotent neurospheres prepared after passage of primary transgenic neurospheres derived from all three of these developmental stages. In addition, GCV prevented growth of multipotent neurospheres from transgenic astrocyte-enriched cell cultures derived from postnatal GZ, and elaidic acid GCV given for 4 d to adult transgenic mice in vivo abolished the ability to grow multipotent neurospheres from GZ. Extensive control experiments, including clonal analysis, demonstrated that failure of neurosphere growth was not merely secondary to loss of GFAP-expressing support cells or the result of a nonspecific toxic effect. Our findings demonstrate that the predominant multipotent NSCs isolated from postnatal and adult but not early embryonic GZs express GFAP, and that NSCs exhibit heterogeneous expression of intermediate filaments during developmental maturation.
发育中和成年大脑的脑室周围生发区（GZs）含有神经干细胞（NSCs），但其细胞身份和起源尚未完全明确。我们使用组织培养技术和从鼠胶质纤维酸性蛋白（GFAP）启动子表达单纯疱疹病毒胸苷激酶（HSV-TK）的转基因小鼠，来检验某些神经干细胞表达GFAP这一假说。为此，我们确定了在不同发育阶段由源自GZs的GFAP表达细胞形成的多能神经球的相对比例。在这个转基因模型中，通过用抗病毒药物更昔洛韦（GCV）处理，选择性地消除了分裂的GFAP表达细胞。单细胞分析表明，转基因衍生的HSV-TK仅存在于GFAP表达细胞中。体外应用GCV消除了出生后和成年转基因小鼠GZs中多能神经球的生长，但未消除早期胚胎（胚胎第12.5天）转基因小鼠的多能神经球生长。GCV阻止了从所有这三个发育阶段衍生的原代转基因神经球传代后制备的二代多能神经球的生长。此外，GCV阻止了出生后GZ来源的富含转基因星形胶质细胞的细胞培养物中多能神经球的生长，并且在体内给成年转基因小鼠连续4天给予反式油酸GCV消除了从GZ生长多能神经球的能力。广泛的对照实验，包括克隆分析，表明神经球生长失败不仅仅是由于GFAP表达支持细胞的丧失或非特异性毒性作用的结果。我们的研究结果表明，从出生后和成年而非早期胚胎GZs分离的主要多能神经干细胞表达GFAP，并且神经干细胞在发育成熟过程中表现出中间丝的异质表达。
